Propagation and/or derivation of embryonic stem cells

ABSTRACT

Embryonic stem (ES) cells are cultured in the presence of a compound which selectively inhibits propagation or survival of cells other than ES cells. The ES cells have not been genetically altered. Instead, the compound inhibits a signalling pathway which is essential for propagation of differentiated cells but is not essential for propagation of ES cells—hence ES cells are selectively maintained in the culture.

The present invention relates to propagation and/or derivation ofembryonic stem (ES) cells and to compositions therefor.

Stem cell self-renewal underpins growth and diversification duringdevelopment of the mammalian embryo and tissue repair and homeostasis inthe adult. However, studies on stem cell biology have been hampered bythe absence of normal, non-transformed stem cells that can be propagatedin vitro. An exception to this are mouse ES cells, which can be culturedindefinitely as pluripotential stem cells when the medium issupplemented with a ligand that activates the cytokine receptor gp130.These stem cells are present only transiently in the early embryo.However, they are intrinsically tumorigenic and give rise to stem celltumours, teratocarcinomas, when early embryos are grafted to ectopicsites. Furthermore, when the epiblast of a mouse blastocyst is explantedin culture, immortal embryonic stem (ES) cell lines can be derived.

Propagation of ES cells is dependent on the presence of the cytokineLIF, which promotes the proliferation of undifferentiated stem cellsthrough the activation of a heteromeric complex containing two relatedcytokine receptors, gp130 and the low affinity LIF receptor, LIF-R.

Signal transduction via gp130 depends upon the activation of JAKkinases, a class of non-receptor tyrosine kinases that associate withthe membrane proximal box1/box2 region of cytokine receptors. Uponactivation, JAKs phosphorylate tyrosines in the intracellular domain ofgp130 creating binding sites for proteins containing Src-homology-2(SH2) domains. These proteins can in turn be phosphorylated, resultingin the activation of a variety of signalling molecules, including STATs(signal transducer and activator of transcription) 1 and 3, the tyrosinephosphatase SHP-2, the mitogen activated protein kinases ERK1 and ERK2,insulin receptor substrate-1 (IRS-1), Grb2 associated docking protein(Gab1) and phosphatidylinositol (Pl)-3 kinase and the non-receptortyrosine kinases hck and btk. Amongst these, both the STAT and MAPKsignalling pathways have been demonstrated to play essential roles inmediating the biological responses to ligands that activate gp130 invarious cell types.

STATs are a family of latent transcription factors that upon recruitmentto a receptor become phosphorylated, dimerise and then translocate tothe nucleus where they regulate transcription of target genes. We haverecently shown that activation of STAT3 is required for maintaining thepluripotent phenotype of ES cells. Chimeric gp130 receptors unable toengage STAT3 were incapable of signalling self-renewal, whilstover-expression of a STAT3 interfering mutant caused ES cells todifferentiate. However, in the absence of constitutively active forms ofSTAT3, we have been unable to determine whether this regulator alone issufficient or if other signals are also required for self-renewal.

gp130 can also associate with the protein tyrosine phosphatase. Thiswidely expressed enzyme has also been implicated in signal transductionfrom receptor tyrosine kinases (RTKs) and is regarded as a positiveeffector of the ERK signalling cascade. Although the biologicallyrelevant substrates for SHP-2 phosphatase have not been unequivocallyidentified, it is significant that over-expression of catalyticallyinactive SHP-2 mutants can suppress receptor mediated activation of theERK pathway. Recruitment to the gp130 receptor complex also results intyrosine phosphorylation of SHP-2. These phosphotyrosines can serve asbinding sites for the adaptor protein Grb2, which potentially couplesthe receptor, through interactions with SOS and Ras, to the ERK pathway.Stimulation of ERK1 and ERK2 has been demonstrated to play a role inmediating mitogenic responses of cells to growth factors, though theprecise nature of this role is yet to be elucidated.

It is known to maintain cultures of ES cells in the presence of certainfactors that specifically promote proliferation of ES cells, and LIF isone such factor. This does not, however, absolutely preventdifferentiation; there is a continuing loss of ES cells from thesecultures which accordingly in time become overgrown with differentiatedcells. Thus, it remains a problem to reduce even the small rate ofdifferentiation of ES cells when propagated in such known factors.

A further problem in this art is that the range of ES cells that can bepropagated in culture is limited to just a few types, mainly mouse EScells, despite efforts to derive ES cells from other species.

It is also known to introduce into ES cells a selectable marker that isdifferentially expressed in (i) ES cells and (ii) cells other than EScells. Selection can then be used to eliminate those cells that havedifferentiated. But, this requires genetic alteration of the ES cells.

An object of the invention is to provide an alternative method ofobtaining and/or culturing ES cells. Another object of the invention isto reduce the rate of differentiation of ES cells in known cultures. Afurther object is to provide culture medium components for maintenanceor derivation of a culture of ES cells.

The present invention is based upon the discovery of a class ofcompounds that selectively promote self renewal of ES cells and/orinhibit propagation or survival of cells other than ES cells, ieselectively acting on differentiated cells.

Accordingly, the present invention provides a method of culture ofembryonic stem (ES) cells, comprising maintaining the ES cells in thepresence of a compound which selectively inhibits propagation orsurvival of cells other than ES cells.

It is an advantage that the invention uses a compound that acts ondifferentiated cells and enables selective removal or killing orretardation of growth of cells that have differentiated, thusfacilitating retention of a relatively pure culture of ES cells.Previously, use of known ES propagating factors, say, LIF, resulted in acertain low level of differentiation of ES cells. This level can furtherbe reduced according to the invention, using both the known factor andthe compound of the invention.

The effect of the compound of the invention is selective in that itsinhibitory effects are seen to a greater extent on differentiated cellsthan on ES cells. It is preferred that the compound has substantially noinhibitory effect on ES cells.

Preferably, the compound inhibits a signalling pathway which whenactivated or at least maintained, ie not inhibited, leads to or enablespropagation of cells other than ES cells. In this way the compound iseffectively selectively toxic to the differentiated cells compared withits effect, if any, on ES cells. Differentiated cells either die or havetheir growth slowed in its presence. Inhibition may be total or partialand may occur at different points along the pathway and any compoundthat has the effect of inhibiting the pathway is to be regarded as aninhibitor. By reference to signalling pathway it is intended to includepathways in which an endogenous or exogenous substance has a directeffect upon cell function, e.g. propagation, cell division, the cellcycle, metabolism, as well as via an indirect effect such as viareceptor-mediated signalling pathways.

A further advantage of the invention is that selection of ES cells andtheir maintenance in culture is achieved without the need for geneticmanipulation of the cells. This represents a major advantage inparticular in connection with derivation of ES cells from humans.Instead, in preferred embodiments of the invention selection and/orderivation of ES cells is achieved by the discovery of a signallingpathway, which pathway can be blocked or inhibited, activation ormaintenance of which is essential for propagation of differentiatedcells but not for propagation of ES cells. By “essential” it is meantthat the differentiated cell is at least severely handicapped, such ashaving its growth severely retarded or other fundamental cellularfunctions severely adversely affected, when the pathway is inhibited aswell as meaning that cellular growth stops or the cell dies when thepathway is inhibited. If inhibition of the pathway did not actuallyresult in death of the differentiated cell but instead a relative growthslowing such as to provide selective propagation of ES cells in culturethen the effect desired in the invention is achieved. Preferably,though, differentiated cells have their growth substantially stoppedcompared with ES cells.

In an embodiment of the invention, the compound inhibits or reducesactivity of a component of the ras/MAPK cascade. In use, the inhibitoris present in culture medium at non-toxic levels and inhibits areceptor-mediated pathway which would normally when activated lead topropagation of differentiated cells but which is not needed forpropagation of ES cells. As a result, selective proliferation of EScells is obtained.

In a particular embodiment of the invention, the compound inhibits oneor more mitogen activated protein kinases, for example ERK1 and ERK2. Inanother embodiment of the invention, the compound inhibits SHP-2, forexample by inhibiting binding of the enzyme to gp130, having a similareffect. In a further embodiment, the inhibitor inhibits MEK.

In a specific embodiment of the invention, described in examples below,the MEK inhibitor PD098059 is used to sustain ES cells in culture in anundifferentiated state. A further specific embodiment of the inventionis the mitogen-activated protein kinase kinase inhibitor U0126, which isselective for MEK-1 and MEK-2 (Favata et al, 1998). Anthrax lethalfactor has also been found to exhibit an MAPKK inhibitory profilesimilar to that of PD098059 (Duesbery et al, 1999). These variouscompounds may be used alone or in combination or with other factors.

The inhibitor of the invention may inhibit the cell cycle indifferentiated cells, thereby preventing or slowing cell growth. Aspecific inhibitor of the invention inhibits MEK and induction of cyclindownstream of this enzyme is as a result disrupted; thus the variousphases of the cell cycle are affected, and cyclin dependent entry intoS-phase of the differentiated cells is inhibited. The selectivepropagation of the invention may alternatively be achieved bydownregulation of a component of the ras/MAPK cascade. MKP-3 is anexample of a MAP kinase phosphatase and a known downregulator of theERKs, and in a specific example of the invention MKP-3 has beenintroduced into an ES cell by way of a transgene. It is further anoption for selective culture of ES cells to be obtained through the useof a combination of any two or more of the inhibitors of the invention.Specifically, both a component of the ras/MAPK cascade and SHP-2 may beinhibited concurrently, though generally any combination of inhibitorsis encompassed by the invention.

By “propagation”, and corresponding terms, it is intended to mean thatan ES cell has formed daughter cells so that the total number of EScells is increased, ie the ES cell has survived and multiplied.“Proliferation”, and its corresponding terms, is intended to have thesame meaning. “Self-renewal”, and its corresponding terms, is intendedto mean that at least one daughter cell is identical to the parent.

According to a second aspect of the invention there is provided a methodof culture of ES cells comprising maintaining ES cells in the presenceof a first compound that promotes proliferation of ES cells and a secondcompound that enhances the response of the cells to the first compound.

This has the advantage that increased ES cell proliferation is achievedfor a given amount of the first compound. In an example of the method,LIF is used to promote ES cell propagation as is known in the art and anamount of the second compound increases the effects of LIF. The firstcompound preferably acts through a cell-surface receptor and exerts itsactivity through at least one receptor subunit, and the second compoundmodifies an intracellular signalling pathway so as to increase theresponse of the ES cell to the first compound.

The second compound may suitably be an inhibitor according to the firstaspect of the invention. The second compound may also bind to orotherwise affect the same receptor subunit that transduces the ES cellgrowth inducing signal from the first compound.

A third aspect of the invention provides a method of culture of ES cellscomprising maintaining ES cells in the presence of:

-   -   (a) a compound that promotes propagation or survival of ES        cells; and    -   (b) a compound that inhibits propagation or survival of cells        other than ES cells.

Compound (a) may be selected from known compounds that promote ES cellproliferation, especially LIF, and used in combination with a compound(b) which may in turn be selected from compounds of the first aspect ofthe invention.

In a preferred embodiment of the invention the combination of compounds(a) and (b) is synergistic. Thus, ES cells are maintained in thepresence of two factors that together increase the percentage of EScells that self renew, and this increase is greater than the combinedincrease in percentage of self renewal when the two compounds are usedin separate cultures.

Compound (b) suitably selectively inhibits a signalling pathwayessential to propagation of cells other than ES cells, leading toselective death or growth inhibition of differentiated cells combinedwith propagation of remaining ES cells as mediated by compound (a). Inan embodiment of the second aspect of the invention compound (b) isselected from the inhibitory compounds described above and below inrelation to the first aspect of the invention, and is preferably aninhibitor of the ras/MAPK cascade. Compound (a) is typically selectedfrom known compounds that promote proliferation of undifferentiated EScells, such as a cytokine that activates the cytokine receptor gp130 inES cells. LIF is one example. Another is a combination of IL-6 andsIL-6R.

The invention additionally provides in a fourth aspect a culture mediumfor culture of ES cells and comprising a compound that selectivelyinhibits propagation or survival of cells other than ES cells.

In a fifth aspect the invention provides a medium for culture of EScells comprising a first compound that promotes proliferation orsurvival of ES cells and a second compound that enhances the response ofthe ES cells to the first compound.

In a sixth aspect the invention provides a culture medium for culture ofES cells and comprising (a) a compound that promotes proliferation orsurvival of ES cells, and (b) a compound that inhibits propagation orsurvival of cells other than ES cells.

The culture media of the invention preferably are characterised by thecomponents as described in relation to the first to third aspects of theinvention and preferably further comprise conventional culture mediaingredients.

The invention still further provides a method of obtaining and/ormaintaining a substantially pure culture of ES cells comprisingculturing ES cells in the presence of culture medium according to any ofthe fourth to sixth aspect of the invention. The invention is ofapplication without limitation to ES cell type, and may suitably beapplied to vertebrate cells, in particular mammalian cells, primatecells, rodent cells, and human cells. In a specific embodiment describedbelow in further detail, mouse cells have been used. By “ES” cells it isintended to encompass embryonic stem cells, embryonic carcinoma cells,embryonic gonadal cells, embryo-derived pluripotential stem cells andgermline-derived stem cells.

A yet further aspect of the invention provides a method of deriving EScells comprising isolating cells from an embryo or embryoid body andmaintaining a culture of those cells in the presence of a compound thatselectively inhibits propagation or survival of cells other than EScells. The cells thereby obtained may then be maintained in,the presenceof the or a further compound that selectively inhibits propagation orsurvival of cells other than ES cells. In a particular embodimentdescribed below, the method comprises developing an embryo in vivo,harvesting the embryo prior to pro-amniotic cavity formation andisolating cells therefrom, and deriving ES cells from the isolated cellsor, alternatively, culturing the embryo in vivo in the presence of thecompound prior to isolating ES cells therefrom.

A yet still further aspect of the invention provides a method ofderiving ES cells comprising developing an embryo in vitro, isolatingcells from the inner cell mass of the embryo and maintaining those cellsin the presence of a compound that selectively inhibits propagation orsurvival of cells other than ES cells. The method may comprise removingprimitive endoderm prior to culture in the presence of the compound.

In the above two aspects of the invention relating to derivation of EScells, the compound that selectively inhibits propagation or survival ofcells other than ES cells is selected from compounds which inhibitactivity of the enzyme SHP-2, inhibit the ras/MAPK cascade, inhibit MEK,inhibit a mitogen activated protein kinase or inhibit cyclin dependententry into S-phase of non-ES cells. Thus is opened the possibility toderive ES cells through the ES cell—selective mechanisms of theinvention, to derive ES cells for the first time for various speciesfrom which this has hitherto not been possible.

In a specific embodiment of the present invention, ES cells werepropagated in vitro whilst retaining the capacity, upon reintroductioninto a blastocyst, to contribute to all cell types of the developinganimal, including the germ line. These cells therefore represent atractable experimental system with which to study the regulation of stemcell self-renewal. The identification of underlying mechanisms thatregulate ES cell propagation should allow the development of improvedstrategies for establishing ES cell lines from other mammalian speciesand may contribute to our understanding of self renewal in somatic stemcells.

Ligand mediated engagement of gp130 in differentiated cells results inthe recruitment and phosphorylation of the STAT3 and SHP-2 signallingmolecules. Stimulation of ES cells through either endogenous gp130 orthe chimeric GRgp(278) receptor increased the tyrosine phosphorylationof SHP-2. This modification was blocked when tyrosine 118 of gp130 wasmutated to phenylalanine, confirming that this single tyrosine isessential for recruitment of SHP-2. In spite of this deficit, themutated receptor was fully capable of directing self-renewal of EScells, proving effective even at low concentrations of activatingligand. Interestingly, ES cells in which both copies of the endogenousSHP-2 gene have been mutated are viable and undergo self-renewal. Sincethe mutant protein carries a deletion of the N-terminal SH2 domain, aregion which is known to bind to gp130 these ES cells might be expectedto show an altered responsiveness to LIF—in fact, the cells exhibitedimpaired capacity to differentiate in vitro.

Since activation of ERK 1 and ERK2 is also associated with stimulationof gp130 in ES cells, we examined the activation of these MAP kinases bythe chimeric receptors. Whereas stimulation of gp130 in ES cellstriggered an increase in ERK1 and ERK2 phosphorylation, no responsecould be detected upon engagement of the Y118F chimeric receptor.Furthermore, treatment of ES cells with inhibitory concentrations of theMEK inhibitor PD098059 did not block but rather appeared to slightlyenhance self-renewal of ES cells. Notably, however, in the absence ofLIF, PD098059 did not prevent ES cell differentiation. These resultsestablish that, in accordance with the invention, the activation of ERK1and ERK2, via either SHP-2 or another pathway such as Shc, is notcritical in maintaining the proliferation of ES cells. Selectivepropagation of ES cells is obtainable due to the relatively greaterimportance of activation of these enzymes in non-ES cells.

This lack of a requirement for gp130-dependent ERK activation may berelated to the quasi-transformed nature of ES cells. An establishedfunction of ERKs in differentiated cells is to regulate the transitionthrough G1/S, at least in part through the induction of cyclin D.However, ES have a very short G1 phase and appear to posses few of theG1 associated control mechanisms (Savatier et al., 1994; Savatier etal., 1996). Furthermore, the reduced dependence on ERK signalling isconsistent with the observation that ES cells continue to proliferate inthe absence of serum, a powerful mitogen and inducer of ERK activity(Johansson and Wiles, 1995). In this way, inhibition of cyclin-dependententry into S-phase in accordance with the invention enables selectivepropagation and/or survival of ES cells.

In vitro differentiation of ES cells is associated with the induction ofG1 cyclin expression, the establishment of a long G1 phase and adecrease in the rate of cell division (Savatier et al., 1996). Thistransition probably reflects the changes that normally occur in theembryo at gastrulation, after the initial rapid expansion of epiblastcells. Interestingly, inhibition of SHP-2 activity during embryonicdevelopment of either xenopus or mice is associated with failure togastrulate normally with defects in the formation of mesodermal celllineages. The differentiation of epiblast cells may therefore representthe point at which embryonic cells first become subservient to normalgrowth control mechanisms. Significantly, this is also the stage atwhich transplanted grafts of embryonic tissue lose the capacity to formmalignant teratocarcinomas.

SHP-2/ERK activation is thus not essential for ES cell proliferation.Our results suggest that tyrosine 118 downregulates the activity of thegp130 receptor, causing a dramatic shift in dose response and prolongedactivation of STAT3 in cells stimulated via the GRgp(Y118F) receptor.Until recently, SHP-2 had primarily been regarded as positive effectorof signalling, either as an adaptor protein or a potential activator ofsrc family kinases. However, a negative regulatory function wassuggested both by its homology to SHP-1, a suppresser of erythropoietinreceptor function, and its interaction with CTLA-4, an inhibitor of theT cell receptor. It has been reported recently that mutation of tyrosine118 increases STAT3 signalling in neuroblastoma and hepatoma cells.Transcription from STAT3 responsive promoter constructs was alsoincreased by overexpression of tatalytically inactive SHP-2 proteins,pointing to the phosphatase as a likely mediator of this effect. Thisconclusion was supported by sustained Phosphorylation of both the Y118Freceptor and its associated JAK kinases. However, overexpression ofcatalytically inactive SHP-2 proteins produced only a slight shift indose response in transfected ES cells (TB, CS unpublished), indicatingthat loss of phosphatase activity of SHP-2 may not be wholly responsiblefor the increased activity of the GRgp(Y118F) receptor.

The non-essential function of gp130-dependent SHP-2 and ERK activationin ES cells further emphasises the pre-eminent role of STAT3 inself-renewal. However, the restricted growth of GRgp(Y118F)transfectants in high concentrations of G-CSF is intriguing since itindicates that excessive signalling can interfere with ES cell growth.Hyperactivation of STAT3 is implicated in this phenotype becausestimulation of the Y118F receptor results in prolonged tyrosinephosphorylation of STAT3 and sustained activation of an endogenous STAT3target gene SOCS3. Furthermore, combined mutagenesis of some of theSTAT3 docking sites can suppress the effect of the Y118F substitution.

Signals further downstream of tyrosine 118 could also influence thegrowth and differentiation of ES cells. The increase in self-renewalobserved on treatment with the MEK inhibitor PD098059 implies that ERKactivation impairs the propagation of ES cells. Interestingly, studiesof gp130-dependent regulation of PC12 cell and astrocyte differentiationhave suggested that activation of the MAPK pathway may antagonizesignals mediated via STAT3. In both cases, reduced MAPK activityresulted in augmented transcription from STAT3 dependent reporterconstructs. In ES cells treated with PD098059, self-renewal was enhancedeven at saturating levels of LIF. This suggests that the effect of theinhibitors of the invention is not simply due to an inhibition of ERKactivity stimulated by LIF but may occur by blocking the actions ofdifferentiation inducers present in serum, or those secreted by ES cellsand their differentiated progeny.

The present invention is now described in specific embodimentsillustrated by drawings in which:

FIG. 1 shows gp130-dependent phosphorylation of SHP-2 in ES cells;

FIG. 2 shows effect of mutating tyrosine 118 on gp130-dependentself-renewal and growth of ES cells;

FIG. 3 shows gp130-dependent phosphorylation of ERK1 and ERK2 in EScells;

FIG. 4 shows effect of the MEK inhibitor, PD098059, on ES cellself-renewal and ERK activation;

FIG. 5 shows effect of PD098059 on ES cell pluripotency;

FIG. 6 shows decay of activated STAT3 following stimulation of gp130 andthe chimeric GRgp130 receptors;

FIG. 7 shows gp130-dependent induction of SOCS-3 gene expression in EScells; and

FIG. 8 shows the effect of an MEK inhibitor on stem celldifferentiation.

In more detail, FIG. 1 shows gp130-dependent phosphorylation of SHP-2 inES cells. ES cells expressing either the GRgp(278) or GRgp(Y118F)chimeric receptors were induced with IL-6 (100 ng/ml plus sIL-6R) orG-CSF (30 ng/ml) for 15 minutes. SHP-2 protein was immunoprecipitatedfrom lysates of unstimulated or stimulated cells, fractionated on aSDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Thefilter was probed with anti-phosphotyrosine antibody (upper panel),stripped and reprobed with anti-SHP-2 antibody (lower panel). Theposition of tyrosine phosphorylated forms of SHP-2 and two additionalproteins are indicated by arrows.

FIG. 2 shows the effect of mutating tyrosine 118 on gp130-dependentself-renewal and growth of ES cells:

-   -   (A) Stem cell renewal mediated by GRgp(278) and GRgp(Y118F)        chimeric receptors in response to G-CSF. Self-renewal, as        measured by β-galactosidase expression from the Oct-4 locus was        assayed after 6 days in culture with G-CSF (300 fg-30 ng/ml).        Data for two independent clones are represented as means±s.e.m.        for duplicate determinations of triplicate samples normalized        relative to the response with IL-6(100 ng/ml plus sIL-6R).    -   (B) Photomicrographs of X-gal stained, representative colonies        formed by GRgp(278) and GRgp(Y118F) transfectants after 6 days        culture with 300 fg, 30 pg and 30 ng/ml of G-CSF.    -   (C) Photomicrographs of representative colonies formed by        GRgp(278) and GRgp(Y118F) transfectants after 6 days culture        with no cytokine, IL-6 (100 ng/ml+sIL-6R), G-CSF(30 ng/ml) or,        IL-6 (100 ng/ml+sIL-6R) and G-CSF(30 ng/ml).

FIG. 3 shows gp130-dependent phosphorylation of ERK1 and ERK2 in EScells. ES cells expressing either the GRgp(278) or GRgp(Y118F) chimericreceptors were either untreated or stimulated with IL-6(100 ng/ml plussIL-6R) or G-CSF (30 ng/ml) for 10 or 20 minutes. Cell lysates wereseparated on a 10% SDS-acrylamide gel, electroblotted onto anitrocellulose membrane and probed sequentially with antibodies specificfor the active phosphorylated form of ERK and STAT3. Reprobing thestripped filter with an antibody that binds to both phosphorylated anddephosphorylated ERKs verified that equivalent amounts of protein wereloaded in the samples.

FIG. 4 shows the effect of the MEK inhibitor, PD098059, on ES cellself-renewal and ERK activation:

-   -   (A) Self-renewal of ES cells treated with PD098059. D027 ES        cells grown at a subsaturating level of LIF (5 U/ml) were        treated with PD098059 for 5 days and assayed for β-galactosidase        expression from the Oct-4 locus. Data are means±s.e.m for        duplicate determinations of triplicate samples normalized        relative to the response to LIF.    -   (B) PD098059 dependent inhibition of ERK activation. GRgp(278)        transfected D027 cells were cultured with a subsaturating level        of LIF (5 U/ml) and PD098059 for 48 hours. Cells were then        stimulated with G-CSF (30 ng/ml) for 10 minutes, lysed in sample        buffer and analyzed for ERK activation by immunoblotting with        phosphospecific anti-ERK antibodies. Subsequent probing of the        filter with an antibody that binds to both phosphorylated and        dephosphorylated ERKs confirmed that equivalent amounts of        protein were loaded in all samples.    -   (C) Effect of PD098059 on the dose response of ES cells to LIF.        The dose response of D027 ES cells to LIF in 25 μM PD098059 or        vehicle (0.05% DMSO) was measured by, β-galactosidase expression        from the Oct-4 locus. Data are means±s.e.m for duplicate        determinations of triplicate samples normalized relative to the        maximum response of cells to treatment with LIF(111 U/ml) plus        vehicle.

FIG. 5 shows the effect of PD098059 on ES cell pluripotency. ZIN40 EScells were treated with 25 μM PD098059 plus 5 U/ml LIF for 48 hours,re-fed with medium containing LIF for a further 24 hours and thenmicroinjected into C57BL/6 blastocysts. Embryos were collected at day9.5 of pregnancy and stained for β-galactosidase activity.Representative embryos are shown in the panel.

FIG. 6 shows decay of activated STAT3 following stimulation of gp130 andthe chimeric GRgp130 receptors. GRgp(278) and GRgp(Y118F) ES celltransfectants were stimulated with IL-6 (100 ng/ml plus sIL-6R) or G-CSF(30 ng/ml) for 25 minutes (0*), re-fed with cytokine free medium andsamples were collected at 40 minute intervals. Immunoblots of celllysates were probed sequentially with an antibody specific for theactive phosphorylated form of STAT3 and then with an antibody thatrecognizes both phosphorylated and unphosphorylated STAT3. Note thatactivation of STAT3 is associated with the appearance of a slowermigrating STAT3 species, presumed to be the serine phosphorylated formof STAT3.

FIG. 7 shows gp130-dependent induction of SOCS-3 gene expression in EScells.

-   -   (A) Northern analysis was performed on total RNA (10 g) prepared        from ES cells expressing the GRgp(278), GRgp(Y126-275F) and        GRgp(Y118F) receptors, unstimulated (−) or stimulated either        with LIF (L, 100 units/ml) or G-CSF (G, 30 ng/ml) for the        indicated times (minutes). Hybridisation of the ≈3 kb SOCS-3        mRNA and ethidium bromide staining of the 18S rRNA is shown in        the upper and lower three panels, respectively.    -   (B) Graphic representation of SOCS-3 mRNA expression shown in        panel A. SOCS-3 mRNA hybridization was quantitated by        phosphorimage analysis and signals were normalized relative to        those obtained in each cell line at 90 minutes after stimulation        with LIF.

FIG. 8 shows that an MEK inhibitor of the invention sustainsundifferentiated ES cells in an aggregate culture.

EXAMPLE 1

Materials and Methods

Cell Culture and Transfection

ES cells were maintained without feeder cells in Glasgow modification ofEagles medium (GMEM) containing 10% fetal calf serum, 0.1 mM2-mercaptoethanol and LIF. D027 cells have both copies of the lif geneinactivated by homologous recombination and an IRES-βgeo reporter geneinserted within the Oct-4 gene locus. ZIN40 cells carry a nuclearlocalised β-galactosidase marker gene that is widely expressed indifferentiated cell types. For transfections, 2×10⁷ cells wereelectroporated with 100 μg of linearised plasmid DNA at 0.8 kV and 3 μFin a 0.4 cm cuvette using a Bio-Rad gene pulser. Stably transfectedclones were selected in medium containing 20 μg/ml zeocin (Invitrogen).

Plasmid Construction

The GR/gp130 chimeric receptors were generated by fusing theextracellular domain of the human G-CSFR to an EcoRI fragment of mousegp130 containing the transmembrane domain and the entire cytoplasmicregion. The phenylalanine substitution of tyrosine 118 was introducedinto the intracellular domain of gp130 by PCR overlap mutagenesis. ThePCR product was substituted into the GRgp(278) chimaera and sequenced.The receptor cDNAs were inserted within expression vector pCAGIZ. Thisvector contains a bicistronic expression cassette, consisting ofcytomegalovirus enhancer-human β-actin promoter, a site for insertion ofthe receptor cDNA, an internal ribosome entry site (IRES) and the zeocinresistance gene.

Self-Renewal Assay

Expression of β-galactosidase from the Oct-4 locus in D027 cells wasquantitated in an ONPG assay. Cells were plated at 5000 per well in a24-well dishes and cultured for 6 days in the presence or absence ofcytokine. When cells were treated with PD098059, they were plated at2500 per well and cultured overnight in normal growth medium prior toaddition of the inhibitor. On day 6, cells were washed once with PBS andlysed in 0.4 ml of 0.25 M Tris pH 7.5, 0.5 mM DTT, 0.5% NP40. Lysate (40μl) was mixed with 100 μl of ONPG buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄,10 mM KCl, 1 mM MgCl₂, 50 mM 2-mercaptoethanol, 1.2 mM ONPG) in amicrotitre plate, incubated at 37° C. for 2-4 hours and the absorbancewas read at 420 nm. All assays were performed in triplicate.

Immunoprecipitation and Immunoblotting

One day after plating (2-3×10⁶ cells per 100 mm dish), ES cells werere-fed with medium containing 1% foetal calf serum and lackingcytokines. The following day, cells were transferred to serum freemedium for 4 hours prior to stimulation with IL-6 (100 ng/ml plussoluble receptor) or G-CSF (30 ng/ml) for 15 minutes. Cells were thenwashed once with ice-cold PBS and scraped off in 1 ml of ice-cold lysisbuffer (150 mM NaCl, 10 mM Tris.HCl pH 7.4, 0.5% NP40, 1 mM NaVO₃, 1 mMEDTA, 0.5 mM PMSF). Cleared lysates were incubated with 1_μg anti SHP-2antibody (Santa Cruz) at 4° C. for 1 hour and then protein A sepharosewas added and the incubation continued overnight. Immunoprecipitateswere lysed in 2× SDS sample buffer, fractionated by electrophoresis on a10% SDS polyacrylamide gel and electroblotted onto nitrocellulose. Afterovernight treatment with blocking buffer (25 mM Tris-HCl pH 7.4, 2.7 mMKCl, 140 mM NaCl, 0.1% Tween 20, 1% BSA) the membranes were probedsequentially with anti-phosphotyrosine antibody 4G10 (TransductionLaboratories) and anti-SHP-2 antibody. Blots were incubated with horseradish peroxidase coupled anti-rabbit IgG and developed using ECLreagents (Amersham). Antibodies were stripped from the membranes betweenprobings by incubation at 50° C. for 30 minutes in 62.5 mM Tris.HCl pH6.8, 2% SDS, 100 mM 2-mercaptoethanol.

For analysis of STAT3 and ERK phosphorylation, 1×10⁶ ES cells wereplated per well of 6-well dishes. Cells were serum starved and treatedwith cytokines as described above and then lysed in 100 μl SDS samplebuffer. Ten microlitre aliquots were fractionated on a 10% SDSpolyacrylamide gel, electroblotted onto nitrocellulose and probed withanti-ERK and anti-STAT3 antibodies according to the directions providedby supplier (New England Biolabs).

Northern Blotting

RNA was prepared as described, except that the second iso-propanolprecipitation was replaced by an overnight precipitation at 4° C. in 2 MLiCl to remove contaminating DNA. Total RNA (10 μg) was separated on a0.66 M formaldehyde/agarose gel and transferred to a nylon membrane(Boehringer). Hybridisation was performed as described, using a[α-32P]dCTP labelled DNA probe. The probe was an EcoRI-NotI fragment ofa SOCS3 EST plasmid (IMAGE clone number 864805, Genbank accession numberAA444828 obtained from HGMP). Sequence analysis verified that the probecorresponded to nt 1666-2176 of SOCS-3 (U88328).

Chimaera Analysis

ZIN40 ES cells were injected into C57BL/6 blastocysts and transferredinto pseudopregnant mice. Mice were sacrificed at day 9.5 of pregnancyand the embryos were stained with X-gal.

Results

Tyrosine 118 is Required for gp130-Dependent Phosphorylation of SHP-2 inES Cells

Previous studies in BAF pro-B cell lines have shown that gp130-dependentactivation of SHP-2 and the MAPKs, ERK1 and ERK2, is mediated viatyrosine 118 (located 118 amino acid residues from the membrane) in thecytoplasmic region of gp130. To examine the functional role of tyrosine118 in ES cells, we constructed cDNAs encoding chimaeric receptorsconsisting of the extracellular domain of the granulocyte colonystimulating factor receptor (G-CSFR) fused to the transmembrane andcytoplasmic region of gp130. Since ES cells do not normally express theG-CSFR and show no self-renewal response to G-CSF (data not shown),these chimaeric receptors can be used to examine signaling independentlyof endogenous cytokine receptors. cDNAs encoding either the unmodifiedchimaeric receptor, GRgp(278), or a mutated receptor in whichphenylalanine had been substituted for tyrosine 118, GRgp(Y118F), werecloned into the pCAGIZ expression vector and stably introduced into D027ES cells by electroporation.

In several differentiated cell types, SHP-2 becomes tyrosinephosphorylated following its recruitment to a tyrosine phosphorylatedgp130 receptor subunit. To examine whether SHP-2 undergoes thismodification in ES cells, SHP-2 immunoprecipitates were prepared fromGRgp(278) and GRgp(Y118F) transfectants following stimulation witheither IL-6 (plus sIL-6R) or G-CSF and probed for phosphotyrosine bywestern blotting (FIG. 1A). An increase in phosphorylated SHP-2 wasdetected in cells stimulated through either the endogenous gp130 or theGRgp(278) receptors. Two additional tyrosine phosphoproteinsco-precipitated with the phosphorylated SHP-2. The band migrating atapproximately 100 kD may represent Gab 1, the IRS-1-related adaptorprotein previously reported to associate with phosphorylated SHP-2. Noincrease in phosphorylation of SHP-2 was detected following stimulationof the GRgp(Y118F) receptor, confirming that tyrosine 118 is essentialfor effective gp130-dependent phosphorylation of this phosphatase in EScells.

SHP-2 Activation is Not Required for ES Cell Self-Renewal

In order to determine whether activation of SHP-2 is necessary for thepropagation of ES cells, the response of GRgp130 transfectants to G-CSFwas measured in a self-renewal assay. D027 cells have a LacZ geneinserted within the stem cell specific gene, Oct-4. As a consequence,expression of this integrated reporter gene is restricted toundifferentiated ES cells and the resulting β-galactosidase activityprovides a measure of stem cell self-renewal. In addition, both copiesof the LIF gene have been inactivated through gene targeting, thusreducing autocrine stimulation of ES cell growth. β-galactosidaseactivity from two independently isolated clones for each receptorconstruct was measured in medium density cultures after 6 days oftreatment with 300 fg-30 ng/ml G-CSF.

The data presented in FIG. 2A shows that self-renewal of GRgp(278)transfectants increased in a dose dependent manner, reaching a plateauat 3-30 ng/ml G-CSF. In contrast, the maximal self-renewal response ofGRgp(Y118F) ES cells was achieved at just 30 pg/ml G-CSF. The morphologyof the GRgp(Y118F) colonies maintained in 30 pg/ml G-CSF was typical ofundifferentiated ES cells (FIG. 2B). This result establishes thatactivation of SHP-2 through tyrosine 118 is not required to direct EScell self-renewal. Equivalent levels of both receptor chimaeras wereexpressed at the cell surface of ES cell transfectants, as judged bybinding studies with ¹²⁵I-labelled G-CSF (data not shown). Therefore theshift in dose response suggests that the mutant receptor may haveenhanced signaling activity.

Interestingly, at higher concentrations of G-CSF, GRgp(Y118F)transfectants formed small aggregates of cells rather than the moreflattened colony morphology normally associated with undifferentiated EScells (FIG. 2B). These colonies expressed β-galactosidase and stainedpositive for the stem cell marker alkaline phosphatase (FIG. 2B and datanot shown), indicating that the ES cells remained undifferentiated. Thiswas confirmed by the resumption of typical ES cell growth and colonymorphology when, following the initial treatment with G-CSF, thesecultures were refed with medium containing IL-6 plus sIL-6R (data notshown).

The unusual appearance of GRgp(Y118F) cells in high concentrations ofG-CSF is unlikely to be simply due to an increase in affinity of theY118F receptor for G-CSF because this response is not observed in wildtype cells treated with high levels of LIF, or in GRgp(278)transfectants treated with saturating levels of IL-6(plus sIL-6R),G-CSF, or IL-6(plus sIL-6R) plus G-CSF (FIG. 2C). Furthermore thephenotype of GRgp(Y118F) cells in high levels of G-CSF was maintainedwhen cells were simultaneously stimulated with G-CSF and IL-6 (plussIL-6R). This observation excludes the explanation that the unusual EScell morphology is due to a partial loss of self-renewal signals andsuggests that the phenotype arises from hyperactivation of signalsdownstream of gp130. Collectively these data point to a key role fortyrosine 118 in downregulating gp130 signaling in ES cells.

Tyrosine 118 is necessary for activation of ERK1 and ERK2

Since activation of SHP-2 may couple gp130 to the ERK pathway, weexamined whether tyrosine 118 was also required for activation of ERK1and ERK2 in ES cells. Activation of ERKs in GRgp130 transfectantstreated with G-CSF or IL-6 (plus sIL-6R) was assessed by immunoblottingwith an antibody specific for the phosphorylated (activated) forms ofERK1 and ERK2 (FIG. 3). Basal levels of activated ERK were consistentlydetected in untreated cells following serum starvation. Increased ERKphosphorylation was observed in cells stimulated via the endogenousgp130 and GRgp(278) receptors. This was not evident on stimulationthrough the GRgp(Y118F) chimaera. Reprobing with an antibody specificfor the tyrosine phosphorylated form of STAT3, confirmed that bothchimaeric receptors were effective at activating STAT3. These resultsestablish that tyrosine 118 mediates activation of the ERK pathway in EScells.

Blocking ERK Activation With PD098059 Does Not Impair ES CellPropagation

The capacity of GRgp(Y118F) to signal self-renewal implies that ERKactivation is not required for the propagation of ES cells. To test thishypothesis, D027 cells were cultured in the presence of the specific MEKinhibitor, PD098059. A sub-saturating concentration of LIF (5 U/ml) wasused in these experiments to increase the sensitivity of the assay tochanges in self-renewal signaling. Surprisingly, treatment of ES cellswith 3-25 μM PD098059 did not inhibit self-renewal when compared withcells cultured in vehicle alone (FIG. 4A). More surprisingly, in fact,the level of self-renewal increased in a dose dependent manner with themaximum level being achieved at 12-25 μM. At concentrations greater than50 μM PD098059 the growth of ES cells was impaired, possibly as a resultof some non-specific inhibitory effect of the drug, resulting in smallundifferentiated colonies which stained positive for β-galactosidase byX-gal staining (data not shown).

To verify that ERK activation through gp130 was continuously suppressedby PD098059 in these long-term cultures, GRgp(278) cells were incubatedfor 48 hours with the inhibitor plus LIF and then stimulated through thechimaeric receptor with G-CSF. The immunoblot revealed that G-CSFdependent phosphorylation of ERK1 and ERK2 was progressively reducedfrom 3-12 μM, and effectively blocked at 25 μM PD098059 (FIG. 4B). Thecontinued proliferation of undifferentiated ES cells at inhibitoryconcentrations of PD098059 confirms that gp130-dependent activation ofERK1 and ERK2 is not required for the propagation of ES cells.

The effect of PD098059 on self-renewal suggested that the inhibitormight alter the dose response of ES cells to LIF. Self-renewal of EScells was assayed following treatment with 0.1-100 U/ml LIF either inthe presence of 25 μM PD098059 or vehicle (0.2% DMSO) (FIG. 4C).Treatment with PD098059 increased the level of β-galactosidase activityat all concentrations of LIF. This implies that the drug does not alterthe dose dependency of ES cells but rather enhances their response toLIF. Significantly, PD098059 did not block the differentiation of EScells in the absence of LIF.

ES Cells Propagated in PD098059 Remain Pluripotent

ES cell colony morphology and Oct-4 expression are reliable indicatorsof the undifferentiated phenotype, but do not establish that the cellsare pluripotent. We therefore determined whether ES cells propagated inthe absence of gp130-dependent ERK signaling have the capacity toincorporate into the developing embryo and differentiate appropriately.Cells were cultured at low density (1000 cells/cm²) for 48 hours in thepresence of LIF plus 25 μM PD098059, or in the absence of LIF. They werethen re-fed with medium containing LIF but lacking the inhibitor for afurther 24 hours before microinjection into mouse blastocysts. ZIN40cells were used in this experiment, since they carry a nuclear localisedβ-galactosidase marker that is widely expressed in differentiated celltypes. Staining of mid-gestation embryos for β-galactosidase revealedthat ES cells treated with PD098059 contributed to chimaeras (FIG. 5).However, cells cultured in the absence of LIF for 48 hours wereincapable of colonizing the embryo (data not shown). This resultconfirms that gp130-dependent ERK activity is not required formaintaining the pluripotency of ES cells.

Attenuation of the STAT3 Signal is Mediated Via Tyrosine 118

We have previously established that activation of STAT3 is essential forgp130-dependent self-renewal of ES cells. To determine whether mutatingtyrosine 118 affects this key regulator, activation of STAT3 wascompared in GRgp(278) and (Y118F) transfectants. The acute stimulationof cells for 25 minutes with 30 fg/ml to 300 ng/ml of G-CSF did notreveal a significant difference between the levels of tyrosinephosphorylation of STAT3 induced by the chimaeric receptors (data notshown). However, signaling through the receptors was distinguished whenthe duration of the STAT3 signal was examined (FIG. 6). Cells werestimulated with either G-CSF or IL-6 (plus sIL-6R) for 25 minutes,re-fed with cytokine free medium and then samples were collected at 40minute intervals. A similar time course for the decay of phosphorylatedSTAT3 was obtained following stimulation through either the endogenousgp130 receptor or GRgp(278), with the signal being undetectable at 120minutes. In contrast, the activation of STAT3 was sustained in G-CSFtreated GRgp(Y118F) cells and could still be detected at 160 minutes.This result indicates that tyrosine 118 mediates a signal that normallyattenuates the activation of STAT3.

Substitution of Tyrosine 118 Leads to Hyperinduction of a ChromosomalTarget Gene

In order to investigate whether the prolonged activation of STAT3influenced gene regulation in ES cells, we examined the expression ofSOCS genes. These genes are rapidly induced by cytokines and encodeproteins that can function as negative regulators of cytokine receptorfunction. SOCS-1 is a STAT3 target in Ml cells but this may not be thecase in ES cells as we have not observed any increase in SOCS-1expression in response to LIF (data not shown). In contrast, expressionof SOCS-3 was transiently induced in ES cells stimulated either throughthe LlFR/gp130 complex with LIF or through the GRgp(278) chimaera (FIG.7). The peak level of observed expression occurred at 90 minutes afteraddition of cytokine, and returned close to uninduced levels by 3 hours.There was no induction of SOCS-3 transcripts in ES cells stimulatedthrough a chimaeric receptor, GRgp(Y126-275F), in which the four STAT3docking sites have been eliminated by site directed mutagenesis. Thisresult implies that the SOCS-3 gene is a target for STAT3 in ES cells.Significantly, following activation of the GRgp(Y118F) receptor the peaklevel of SOCS-3 expression obtained at 90 minutes was enhanced and incontrast to stimulation through the LIF receptor or GRgp(278), SOCS-3mRNA levels remained elevated until at least 6 hours post-stimulation.It seems likely therefore that the prolonged activation of STAT3 resultsin enhanced expression of its target genes. This may underlie the shiftin dose response to G-CSF observed for GRgp(Y118F) transfectants.

MEK Inhibitor PD089059 Sustains Undifferentiated ES Cells in AggregateCulture.

Aggregation induces ES cells to differentiate and form structures knownas embryoid bodies that contain multiple differentiated cell types.Undifferentiated cells are largely or wholly eliminated during embryoidformation due to induced differentiation and/or apoptosis.

IOUD2 ES cells, which carry a targeted integration of βgeo into the Oct4locus were used to enable visualisation of undifferentiated cells byhistochemical staining for β-galactosidase. Aggregates were formed inhanging drops by seeding 100 cells/20 μl drop in the presence of 0, 25,50, 75 or 100 μm PD089059. Aggregates were maintained for 6 days, thentransferred to gelatin-coated dishes and allowed to attach overnight.Cultures were then fixed and stained for β-galactosidase activity. Inthe absence of the Mek inhibitor, the embryoid bodies were welldifferentiated and very few Oct4 β-galactosidase expressing cells werepresent in the outgrowths (FIG. 8, upper panel). In the presence ofPD089059 however, the representation of undifferentiated β-galactosidasepositive cells increased in a dose dependent fashion. At PD089059concentrations of 75-100 βm, the great majority of cells wereundifferentiated (FIG. 8, lower panel). The numbers of undifferentiatedcells in these conditions vastly exceeded these present in controlcultures in the absence of MEK inhibitor, therefore this result is notsimply due to ablation of cells This finding indicates that ERKactivation is critical to the process of embryoid body differentiationand that differentiation of stem cells can be prevented by reducing orabolishing Mek activity.

EXAMPLE 2

Derivation of Stem Cell Lines

In order to isolate stem cell lines, embryos were developed in vivo andallowed to implant then harvested prior to pro-amniotic cavity formation(6.5 dpc equivalent). Epiblasts were microdissected and placed insuspension culture in ES cell medium in the presence of PD098059. Afterseveral days in suspension culture the epiblasts were dissociated andplated on tissue culture plastic. PD098059 was maintained in the culturemedium until expanding populations of undifferentiated stem cells weregenerated. Stem cell lines were also derived from embryos developed invitro by immunosurgical isolation of the ICM at the blastocyst stagefollowed by microsurgical removal of the primitive endoderm and culturein ES cell medium plus PD098059.

In accordance with the invention, the independence of ES cellself-renewal from ERK activation has important practical applications.Inhibitors such as of the Ras/MAPK pathway promote the propagation ofundifferentiated ES cells. By suppressing the growth and maturation ofdifferentiated cell types, such inhibitors facilitate the routinemanipulation and de novo derivation of ES cells.

EXAMPLE 3

Expression of MKP-3 Transgene

A transgene coding for MKP-3 was inserted into an ES cell and a cultureof ES cells obtained therefrom expressing MKP-3. It was observed that aculture highly purified in respect of ES cells was maintained and thatdifferentiation of these ES cells was substantially reduced comparedwith differentiation of ES cells in a control culture not expressing theMKP-3 transgene. This experiment also provides genetic evidence insupport of the invention.

REFERENCES

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1-45. (canceled)
 46. A method of maintaining a culture of embryonic stem(ES) cells in a self-renewing state, comprising culturing the ES cellsin the presence of (i) a compound which activates gp130, and (ii) aninhibitor of MEK.